The ultrastructure of hemocytes inDactylopius confusus (Cockerell), and the role of granulocytes in the synthesis of cochineal dye

Summary The ultrastructural study of free circulating hemocytes in the adult cochineal scale,Dactylopius confusus (Cockerell), demonstrated five cell types: prohemocytes, typical granulocytes (T-granulocytes), oenocytoids, plasmatocytes, and granulocytes with modified sub-cellular structure to perform a special synthetic and secretory function, which we refer to as modified granulocytes (M-granulocytes). Prohemocytes showed undifferentiated sub-cellular structure of the basic stem cell type (i.e., high cytoplasmic density with numerous ribosomes, centrally located large nucleus with a distinct nucleolus, and poorly developed endoplasmic reticulum). The commonly observed typical granulocytes (T-granulocytes) had several smooth endoplasmic reticulum (SER) with dilated cisternae and many SER-derived membrane bounded granules of different sizes and electron density. Oenocytoids were identified by the presence of many crystals, RER-originated fine secretory granules, and an eccentric nucleus. Plasmatocytes were easily characterized by their variable shapes and irregular outline with pseudopodia-like cytoplasmic extensions, possession of an elongated lobed nucleus, multivesicular bodies, RER-derived membrane bounded, electron-dense, lysosomelike vacuoles, well-developed SER cisternae, and numerous pinocytic and SER-originated vesicles of different sizes along the peripheral region. M-granulocytes comprised the largest proportion of hemocytes in all samples observed. M-granulocytes were distinguished not only by the presence of membrane bounded granules of different sizes and electron density, but by the possession of large nuclei with distinct nucleoli, many mitochondria, and a highly developed network of rough endoplasmic reticulum (RER). M-granulocytes had abundant, rosette-shaped, RER-derived chains of fine secretory granules, which accumulated in the cytoplasm and vacuoles, and were ultimately deposited into the hemolymph by exocytosis. These fine granules gave a positive result with periodic acid-Schiff (PAS) test. Based on RER-synthesized fine secretory granules (M-granulocytes), their ultimate deposition into hemolymph, the red pigmentation of hemolymph, positive PAS histochemical test of these granules, and the high population of these hemocytes, no such cell type has been described in previous studies in insects. The sub-cellular structure of the granulocyte in this insect has been modified to perform a special synthetic and secretory function (i.e., possibly the synthesis of the red pigment found in hemolymph, which has been the source of commercially important cochineal dye).Abbreviations EM electron microscope - ER endoplasmic reticulum - LM light microscopy - MVB multivesicular body - PAS periodic acid-Schiff - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum - SG secretory granules - TEM transmission electron microscopy - UA uranyl acetate     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Bombyx mori prohemocyte division and differentiation in individual microcultures

We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4-11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae): Effects of parasitism on differential hemocyte counts,micro‐ and ultra‐structures of host hemocytes

Abstract Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) by using only its associated venom, can suppress the immunal responses of Pieris rapae (Lepidoptera: Pieridae). However, up to now, current knowledge of the mechanisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM). Five hemocyte types, prohemocytes (PRs), granulocytes (GRs), plasmatocytes (PLs), oenocytoids (OEs) and coagulocytes (COs), were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.     

红褐斑腿蝗血细胞的形态与分类

利用光学显微镜和显微数码拍照系统,对红褐斑腿蝗Catantops pinguis(Stal)血细胞的形态进行观察和分类。结果在红褐斑腿蝗血淋巴中观察到5种血细胞,分别是原血胞、浆血胞、粒血胞、珠血胞和囊血胞。原血胞为小型圆形细胞,边缘圆滑、清晰,核质比例很大。粒血胞多为中型,形状不规则,边缘凹突不平,内含较大的异质性溶酶体颗粒。浆血胞多为中型,刚离体时形状较规则,常呈圆形、卵圆形。浆血胞内缺乏大的颗粒,细胞核大而圆形,细胞质内具许多小型颗粒状物质。浆血胞离体后形状变化较多,常发展出伪足,呈丝状、短芒状、钩状或片状伪足。珠血胞多为大、中型,外形大体呈圆形,但边缘由于大小不等的珠形内含物突出,呈花瓣状。囊血胞多为中型,圆形或椭圆形,细胞质内具有大小不一的带有折光性的颗粒或块状物,细胞边缘比较光滑。     

华北大黑鳃金龟幼虫血细胞的超微结构观察

应用光学和电子显微镜技术检查了华北大黑鳃金龟3龄幼虫血淋巴内的血细胞,识别出5种类型的血细胞(原血胞、浆血胞、颗粒血胞、珠血胞和凝血胞)并对每一种血细胞的超微结构特点进行描述。     

Classification of larval circulating hemocytes of the silkworm,<Emphasis Type="Italic"> Bombyx mori</Emphasis>, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.     

Fine structure of the hemocytes and nephrocytes of Argas (Persicargas) arboreus (Ixodoidea: Argasidae)

The fine structure of the hemocytes and nephrocytes in Argas (Persicargas) arboreus is described and compared with that of similar cells in other tick species and insects. The hemocytes are of three types: prohemocytes, with a relatively undifferentiated cytoplasm lacking granular inclusions and probably serving as progenitors of the other hemolymph cell types; plasmatocytes, containing abundant mitochondria, cisternae of rough endoplasmic reticulum (RER), and free ribosomes, as well as some small granular inclusions; granulocytes, the predominant cell type in the hemolymph, containing numerous granules of variable electron density and maturity, and pseudopodia-like processes on the cell surface. Plasmatocytes and granulocytes are phagocytic and possibly also have other functions in the tick body. Cells with intermediate features appear to be in a stage of transition from plasmatocyte to granulocyte. Nephrocytes contain vacuoles enclosing fibrillar material, some electrondense granules, and moderate amounts of the active organelles—mitochondria, RER, and ribosomes. The nephrocyte is surrounded by a basal lamina and its plasma membrane infolds to form many deep invaginations coated by a fine fibrillar material. Openings to these invaginations are closed by membranous diaphragms. Coated tubular elements connect the surface invaginations with large coated vesicles, which appear to be specialized for internalization of proteins from the hemolymph. The dense granules may represent an advanced stage of condensation of ingested protein and thus may be lysosomal residual bodies, or they may develop by accumulation of secretory products.     

Circulating hemocytes from larvae of the paper wasp Polistes dominulus (Hymenoptera, Vespidae)

Circulating hemocytes from larval stages of the paper wasp Polistes dominulus were characterized by light and transmission electron microscopy. Three types were identified: prohemocytes, plasmatocytes and granulocytes. The first two are agranular cells while the latter present typical cytoplasmic inclusions called granules. Plasmatocytes differ from prohemocytes being larger, showing lower nucleus/cytoplasm ratio and they possess many phagolysosomes. The substantial uniformity of most subcellular features and the presence of "intermediate forms" support the "single-cell theory" i.e., there is only one cell line that originates from the prohemocyte and leads to the granular cell passing through the plasmatocyte. This hypothesis seems to be confirmed by functional tests. Indeed, most part of cells adheres to the glass and is able to phagocytize fluorescent microspheres.     

Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori

Silkworm (Bombyx mori) hemocytes were fixed immediately after collection. Thin sections of the hemocytes were stained by an indirect immunogold staining method using rabbit anti-prophenoloxidase/IgG as a primary antibody and colloidal gold coated with goat anti-rabbit/IgG as a secondary antibody. Electron micrographs of the sections revealed that only plasmatocytes and oenocytoids have prophenoloxidase both in cytoplasm and nucleus whereas granulocytes, spherulocytes and prohemocytes do not have appreciable amounts of the proenzyme. Cytoplasmic inclusions of oenocytoids also contain the proenzyme. A wide variety of concentrations of prophenoloxidase was observed among oenocytoids. Plasmatocytes appeared to have less prophenoloxidase than any oenocytoids. Once materials in the granules of granulocyte were discharged into the plasma and formed coagula, they cross-reacted with antiprophenoloxidase/IgG, suggesting that prophenoloxidase was trapped in the coagula by unknown mechanisms. This observation is discussed in relation to the dispute concerning the presence of prophenoloxidase or phenoloxidase in the granulocyte.     

Morphology and differential counts of hemocytes of healthy and wound tumor virus-infected Agallia constricta

Six types of hemocytes were found in Agallia constricta leafhoppers: plasmatocytes, spherule cells, granular hemocytes, adipohemocytes, oenocytoids, and prohemocytes. Plasmatocytes, spherule cells, and granular hemocytes accounted for 90–95% of all hemocytes in numphs and adult leafhoppers. As the insect aged from second- and third-instar nymphs to 7- and 8-week-old adults, there was a significant decrease in plasmatocytes in healthy leafhoppers compared to wound tumor virus-infected insects. In contradistinction, there were more granular and spherule hemocytes in healthy leafhoppers than in virus-infected ones as the insects aged. In general, there were more prohemocytes in infected than in healthy leafhoppers. Plasmatocytes from 4- to 8-week-old, infected leafhoppers contained large irregularly shaped, cytoplasmic inclusions. Electron microscopy of these cells showed that the inclusions were either large accumulations of wound tumor virus particles or virus-free electron dense bodies.     

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm,Bombyx mori

Summary A monospecific antibody against -1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

Hemocytes of the centipede Scutigera coleoptrata (Chilopoda,Notostigmophora) with notes on their interactions with the tracheae

The hemocytes of Scutigera coleoptrata were investigated by light and electron microscopy. Four types of hemocytes were identified: prohemocytes, plasmatocytes, granulocytes, and spherulocytes. Only granulocytes could be distinguished from the three other types by May-Grünwald staining, as this is the only hemocyte type demonstrating an eosinophilic reaction. Shape and size give further indications for distinguishing the cell types. In addition, differentiation is possible on the basis of their ultrastructure. However, only a combination of all three methods (staining and light and electron microscopy) allows clear separation of the cell types. As transitional stages between the cell types occur in S. coleoptrata, it is likely that prohemocytes, plasmatocytes, and granulocytes are ontogenetic stages of a single cell lineage. Special cell components and their possible functions are described. Plasmatocytes exocytose tubular structures that probably play a role in coagulation processes. These tubular structures develop in the grana of plasmatocytes. Also, a special arrangement of microtubules and microfilaments was demonstrated. For the first time interactions between hemocytes and tracheae are documented within the Chilopoda. It is assumed that the hemocytes meet their oxygen requirements directly from the tracheae. Phylogenetic implications of the results are discussed.     

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.     

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.     

Fine Structure of the Hemopoietic Tissues in the Mealworm Beetle, Tenebrio molitor

The fine structure of the hemopoietic tissue and its detailed reticular organization in the mealworm beetle, T. molitor were examined using light and scanning electron microscopes. The major hemopoietic tissues in the abdomen were located on the upper surface of the dorsal diaphragm which continuous over the ventral wall of the heart. Histologic characteristics of this hemopoietic tissues are dense clusters of cells. They are irregular in outline and are not surrounded by any connective tissue sheath. The hemopoietic tissue of this insect is consisted of three cellular components which are the reticular cells, hemocytic stem cells and several kinds of mature hemocytes. The reticular cells had numerous cytoplasmic processes and forming a complex network. The stem cells give rise to differentiating hemocytes of the different cell lineages. Mature hemocytes within this hemopoietic tissue are originated from the stem cells and differentiated into several types of hemocytes including prohemocytes, plasmatocytes, and granulocytes.     

家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

The hemocytes of Heliothis armigera: Ultrastructure,functions, and evolution in the course of larval development

Five types of hemocytes, prohemocytes, typical plasmatocytes, coagulocytes, spherule cells, and oenocytoides, have been defined in the last larval instar of Heliothis armigera on the basis of ultrastructural microscopy, smears, and optical phase-contrast microscopy. Modifications in typical plasmatocytes and coagulocytes have been evidenced in the course of development in this instar, which suggests that these hemocytes are involved in physiological processes of development. Only coagulocytes exhibit endocytotic capacities. Phenoloxidase activity was observed in oenocytoides.     

Structural characterization of the hemocytes of Plodia interpunctella

Six types of hemocytes were identified in fifth instars of the Indian meal moth, Plodia interpunctella. The morphology of these cells was characterized by phase contrast and electron microscopy, with Sudan black B, Giemsa, Janus green B, and periodic acid-Schiff staining. Reaction of the hemocytes with seven fluorescing lectin conjugates revealed distinctive binding patterns by their plasma and nuclear membranes and cytoplasmic inclusions. A direct line of descent from prohemocytes to plasmatocytes to granulocytes is suggested from these morphological observations.